Detection units and methods for detecting a target analyte

ABSTRACT

The present application relates to detection units and methods for detecting one or more target analytes in a sample using a complex formed by a target and first and second probes, wherein the complex comprises an elongated region, a particle that is coupled to the first probe, and a solid support that is coupled to the second probe. Specific binding of a target analyte can be distinguished from non-specific binding of the particle by measuring the displacement of the particle.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/217,253, filed Dec. 12, 2018, now allowed; which is a continuation ofU.S. application Ser. No. 15/033,629, filed Apr. 30, 2016, now U.S. Pat.No. 10,179,930; which is 371 of PCT/US2015/014616, filed Feb. 5, 2015;which claimed the benefit of U.S. Provisional Application No.61/936,863, filed Feb. 6, 2014, the contents of which are incorporatedby reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Federal AwardIdentification Number R21CA174594 awarded by the Department of Healthand Human Services/National Institutes of Health/National CancerInstitute. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to detection units and methodsfor detecting a target analyte such as natural, synthetic, modified orunmodified nucleic acids or proteins in a sample.

BACKGROUND OF THE INVENTION

Many detection systems for determining the presence or absence of aparticular target analyte in a sample are known. Examples of detectionsystems for detecting analytes include immunoassays, such as an enzymelinked immunosorbent assays (ELISAs), which are used in numerousdiagnostic, research and screening applications. Generally, thesedetection systems detect the target analyte when it binds to a specificbinding agent or probe resulting in a measurable signal.

When using known detection systems, such as immunoassays, the ability todetect a target analyte is often limited by the low concentration of thetarget analyte in the sample and by non-specific interactions, such asnon-specific binding of signal producing molecules and non-specificbinding of sample molecules. The ability to detect a target analyte in abiological sample is often limited by these two factors.

The signal generated by detection systems is normally proportional tothe number of target analytes that bind to the specific binding probe.Therefore, when the concentration of target is low, the signal is low.The total signal can be increased by increasing the signal associatedwith each bound target analyte. Often, detection systems use a solidsupport and reporter markers, such as fluorescent molecules, to generatethe signal. Several strategies that use reporter markers have beendesigned to increase the signal associated with each bound target, suchas in branched-DNA (Hendricks et al., Am J Clin Pathol. 1995,104(5):537) and hybrid capture (WO 2003078966 A2). While thesestrategies increase the total signal, they often also increase thebackground noise resulting from the non-specific interaction between thereporter marker and the solid support. These strategies do not offer aneffective method of discriminating reporter markers non-specificallybound to the solid support.

The use of micrometer scale particles as reporter markers, described inPCT/GB2010/001913, offers a method to remove particles non-specificallybound to the solid support by applying a controlled fluid drag force onthe particles. However, the drag force significantly reduces the signalas well as the background noise because the disrupting force experiencedby the target containing tethers is as high as the force experienced bythe non-specific tethers.

Another strategy, disclosed in PCT/GB2010/001913 (WO 2011/045570 A2),uses a magnetic bead tethered to a solid support by an elongatedmolecule as a sensing apparatus to detect, for example a signal from anELISA assay. According to this disclosure, the bead is tethered to thesolid support independently of the presence or absence of targetmolecules and the signal is amplified be releasing manipulating agentsthat act on the elongated molecule. This strategy does not provide asimple method to discriminate non-specific interactions.

Accordingly, there is a need for a detection unit and systems of suchunits as well as methods capable of detecting low concentrations oftarget analytes while distinguishing non-specific binding from specificbinding in the sample.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of detecting atarget analyte in a sample comprising:

-   -   a) providing a complex formed from:        -   i) a first probe coupled to a particle and bound to said            analyte if present, and        -   ii) a second probe coupled to a solid support and bound to            said analyte if present, so that if the target analyte is            present in the sample, the particle is indirectly coupled to            the solid support via a complex formed by the target analyte            and the first and second probes, and wherein the complex            comprises an elongated region; and    -   b) either i) applying a force to the indirectly coupled particle        or to the complex comprising the elongated region and measuring        the amount of particle displacement or the length of the        complex, wherein the amount of displacement or length of the        complex indicates whether or not the target analyte is present        in the sample, or ii) measuring the Brownian motion of the        indirectly coupled particle, wherein the amount of Brownian        motion indicates whether or not the target analyte is present in        the sample.

In another aspect, the present invention provides a kit for detecting atarget analyte in a sample, the kit comprising a) a particle; and b) afirst probe capable of binding to said analyte, and to either a solidsupport or to said particle, said first probe optionally comprising anelongated region between about 0.15 and about 20 μm long; c) packagingmaterial; and optionally d) instructions for use.

In yet another aspect, the present invention provides a method ofdetecting a target analyte in a sample, the method comprising:

-   -   a) providing a complex formed from:        -   i) a first probe coupled to a particle and bound to said            analyte if present, and        -   ii) a second probe coupled to a substantially flat solid            support and bound to said analyte if present,    -   so that if the target analyte is present in the sample, the        particle is indirectly coupled to the solid support via a        complex formed by the target analyte and the first and second        probes, and wherein the complex comprises an elongated region;        and    -   b) applying a force to the indirectly coupled particle using a        flow substantially parallel to the solid support that removes        non-specifically bound particles, wherein the presence of the        particle after the application of force indicates the presence        of the target analyte in the sample.

While certain particles (e.g., micrometer scale magnetic beads) can beused to increase the sensitivity of detection systems to generate ameasurable signal, these particles are prone to non-specificinteractions with the solid support to which the probe is attached,creating background noise. However, by using a particle indirectlycoupled to the solid support via a complex formed by the target analyteand the first and second probes, and wherein the complex comprises anelongated region, specific binding to a target analyte can bedistinguished from non-specific binding by measuring the displacement ofthe particle. Particles that are coupled to the solid support via thecomplex are displaced by a distance that is a function of the length ofthe elongated region. Particles that are non-specifically bound to thesolid support are displaced by a distance less than the particles thatare specifically coupled to the solid support via the complex. In thisway, displacement of the particles can be used to distinguish specificfrom non-specific binding, particularly in samples with lowconcentrations of target analyte. Other non-specific interactions thatcan produce the non-specific attachment of a particle to the solidsupport can also be distinguished, such as particles thatnon-specifically bind to a probe, or probes that non-specifically bindto the solid support, because those particles are also displaced by adistance less than the particles that are specifically coupled to thesolid support via a complex.

Using a particle indirectly coupled to the solid support via a complexformed by the target analyte and the first and second probes, andwherein the complex comprises an elongated region in embodiments of thisinvention has at least three additional advantages over other systemsthat use reporter markers. First, using a complex with an elongatedregion creates a simple multiplexing method. Detection of multipletargets in a single assay is possible by using elongated regions ofdifferent size for each different target. Particle displacement andtherefore elongated region length can be easily measured in thousands ofcomplexes with sub-micrometer resolution as demonstrated in Example 3.Second, in some embodiments, the application of force can increase thetarget selectivity of the technique by removing particles that are boundvia molecules in the sample that are similar to but not exactly the sameas the target molecule. Third, in embodiments that apply a forcesubstantially parallel to the solid support, such as embodiments thatapply fluid drag substantially parallel to the solid support, force canremove non-specifically bound particles while not significantly reducingthe signal because being part of a complex with an elongated regionreduces the force experienced by the target analyte. When forcesubstantially parallel to the solid support is applied on particlesbound to the solid support, the tension on the tether decreases withtether length (Langmuir (1996) 12(9): 2271). Therefore, non-specificinteractions, which are normally tethers about 10 nm long, experiencetensions that are significantly higher than the tension that a targetbound in an elongated complex experience. This property of long tethersallows in embodiments of the present invention the removal ofnon-specifically bound particles without significantly affectingspecifically bound particles.

In another embodiment, the method comprises: a) providing a probe and aparticle, wherein the probe comprises a first end for coupling to asolid support, a second end comprising a first analyte binding region,and an elongated region disposed between the first and second end, andwherein the particle comprises a second analyte binding region; b)exposing the probe to the sample, wherein if the target analyte ispresent in the sample, the target analyte binds to the first analytebinding region of the probe; c) exposing the particle to the sample,wherein if the target analyte is present in the sample, the targetanalyte binds to the second analyte binding region of the particle; d)exposing the probe to the solid support, under conditions that permitthe coupling of the first end of the probe to the solid support, so thatif the target analyte is present in the sample, the particle isindirectly coupled to the solid support via the target analyte and theprobe; e) applying a force to the particle; and f) measuring the amountof particle displacement, wherein the amount of displacement indicateswhether or not the target analyte is present in the sample.

In another embodiment, the method for detecting a target analyte in asample comprises: a) providing a probe and a solid support, wherein theprobe comprises a first end for coupling to a particle, a second endcomprising a first analyte binding region, and an elongated regiondisposed between the first and second ends, and wherein the solidsupport comprises a second analyte binding region; b) exposing the probeto the sample, wherein if the target analyte is present in the sample,the target analyte binds to the first analyte binding region of theprobe; c) exposing the probe to the particle, under conditions thatpermit the coupling of the first end of the probe to the particle d)exposing the solid support to the sample, wherein if the target analyteis present in the sample, the target analyte binds to the second analytebinding region of the solid support, so that the particle is indirectlycoupled to the solid support via the target analyte and the probe; e)applying a force to the particle; and f) measuring the amount ofparticle displacement, wherein the amount of displacement indicateswhether or not the target analyte is present in the sample.

In certain embodiments, the first analyte binding molecule of the probecomprises a first nucleic acid that hybridizes to a first region of thetarget analyte and the second analyte binding region comprises a secondnucleic acid that hybridizes to a second region of the target analyte.In certain other embodiments, the first analyte binding region of theprobe comprises a first antibody that binds to the target analyte andthe second analyte binding region comprises a second antibody that bindsto the target analyte.

In yet another embodiment, the method for detecting a target analyte ina sample comprises: a) providing 1) a first probe comprising a first endfor coupling to a particle and a second end comprising a first analytebinding region and 2) a second probe comprising a first end for couplingto a solid support and a second end comprising a second analyte bindingregion, wherein at least one of the first or second probes comprise anelongated region disposed between the first and the second end; b)exposing the first probe and the second probe to the sample, wherein ifthe target analyte is present in the sample, a first region of thetarget analyte binds to the first analyte binding region of the firstprobe and a second region of the target analyte binds to the secondanalyte binding region of the second probe; c) exposing the first probeto a particle under conditions that permit the coupling of the first endof the first probe to the particle; d) exposing the second probe to thesolid support under conditions that permit the coupling of the first endof the second probe to the solid support, so that if the target analyteis present in the sample, the particle is indirectly coupled to thesolid support via the first probe, which is bound to the target analyte,which is bound to the second probe, which is coupled to the solidsupport; e) applying a force to the particle; and f) measuring theamount of particle displacement, wherein the amount of displacementindicates whether or not the target analyte is present in the sample.

In yet other embodiments, the first probe and second probes bind tolocations in the target analyte that are separated by an elongatedregion in the target analyte. In these embodiments the elongated regionin the complex may coincide in part or totally with the elongated regionof the target analyte.

In certain embodiments, the first analyte binding region of the firstprobe comprises a first nucleic acid that hybridizes to a first regionof the target analyte and the second analyte binding region of thesecond probe comprises a second nucleic acid that hybridizes to a secondregion of the target analyte. In other embodiments, the first analytebinding region of the first probe comprises a first antibody that bindsto the target analyte and the second analyte binding region of thesecond probe comprises a second antibody that binds to the targetanalyte.

In certain embodiments, the first probe comprises an elongated nucleicacid disposed between its first and second end, while in otherembodiments, the second probe comprises an elongated nucleic aciddisposed between its first and second end. In yet other embodiments, thefirst probe comprises a first elongated nucleic acid disposed betweenits first and second end and the second probe comprises a secondelongated nucleic acid disposed between its first and second end.

In the methods described herein, the exposing steps (b), (c), and (d),can be performed in any order, or simultaneously. Thus, in certainembodiments, the order of the steps b), c) and d) is b-d-c; c-b-d;c-d-b; d-b-c; or d-c-b. In other embodiments, two out of the three stepsb), c) and d) are conducted simultaneously. In certain embodiments, theexposure of the sample to the first probe is conducted before, after, orsimultaneously with the exposure of the sample to the second probe. Inother embodiments, steps b), c) and d) are conducted simultaneously.

In certain embodiments, the methods comprise a washing step after stepb) and/or step c) and/or step d).

In certain embodiments, the force applied to the particle is a magneticforce, fluid drag, fluid buoyancy, mechanical force, electrical force,centrifugal force, gravitational force, or a combination thereof.

In certain embodiments, the elongated region ranges from about 0.15 μmto about 20 μm in length. In other embodiments, the elongated nucleicacid ranges from about 0.5 μm to about 5 μm in length.

In certain embodiments, the diameter of the particle ranges from about0.3 μm to about 20 μm. In certain other embodiments, the particle is amagnetic particle, including, but not limited to a superparamagneticparticle.

In certain embodiments, instead of measuring the displacement of theparticle generated by the force with or without application of force instep f), the Brownian motion of the particle is measured, wherein theamount of Brownian motion indicates whether or not the target analyte ispresent in the sample.

In certain embodiments, the displacement of the particle is measuredusing an imaging system with a lens,or with a lens-free microscope orwith a coherent imaging technique.

In certain embodiments, the target analyte is a nucleic acid moleculeselected from, single stranded DNA or single stranded RNA such as,messenger RNA, small interfering RNA, micro-RNA and its precursors orcirculating RNA. In certain embodiments, the temperature of the sampleis controlled to produce denaturation of double stranded nucleic acidsin the sample and/or specific hybridization of nucleic acids in thesample to the first and second analyte binding region. In otherembodiments, the sample is initially treated with an exonuclease enzymeto convert double stranded nucleic acids into single stranded nucleicacids. In other embodiments, the target analyte is a protein.

Another aspect of the invention relates to a kit or detection unit foridentifying a target analyte in a sample. The kit or detection unitcomprises a first probe comprising a first end for coupling to a surfaceor a particle, a second end comprising an analyte binding region, and anelongated region disposed between the first and second end. According tothis embodiment, the first probe binds to the target analyte. The firstprobe also allows the amount of displacement of a particle bound theretoto be distinguished from the amount of displacement of a particle boundnon-specifically to a solid support to which the probe is bound. In oneembodiment, the elongated region is a nucleic acid between 0.5 and 10 μmlong. In another embodiment, the kit or detection unit further comprisesa second probe comprising a first end for coupling to a solid support ora particle and a second end comprising a second analyte binding region,wherein if the first end of the first probe is for coupling to a solidsupport, the first end of the second probe is for coupling to theparticle and if the first end of the first probe is for coupling to theparticle, the first end of the second probe is for coupling to the solidsupport.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts an embodiment wherein the first probe (1) and secondprobe comprising an elongated region (2) are exposed to a sample underconditions such that if the target analyte (3) is present in the samplethen it binds to the first probe (1) and the second probe (2). Uponexposure of a particle (4) to the sample, the particle (4) couples tothe first probe (1). Upon exposure to a solid support (5), the secondprobe (2) couples to the solid support (5).

FIG. 1B depicts an embodiment wherein the second probe comprising anelongated region (2) is exposed to a sample under conditions such thatif the target analyte (3) is present in the sample then it binds to thesecond probe (2). Upon exposure of a particle (4) coupled to the firstprobe (1) to the sample, the first probe (1) binds to the target analyte(3). Upon exposure to a solid support (5), the second probe (2) couplesto the solid support (5).

FIG. 2A depicts an embodiment wherein the first probe (1) coupled to aparticle, and the second probe comprising an elongated region (2) areexposed to a sample under conditions such that if the target analyte (3)is present in the sample then it binds to the first probe (1) and to thesecond probe (2). Upon exposure to a solid support (5), the second probe(2) couples to the solid support (5).

FIG. 2B depicts an embodiment wherein the second probe (2) coupled to asolid support (5) and first probe comprising an elongated region (1) areexposed to a sample under conditions such that if the target analyte (3)is present in the sample then it binds to the first probe (1) and to thesecond probe (2). Upon exposure to a particle (4), the particle (4)couples to the first probe (1).

FIG. 3 depicts the effect of the application of force on particlesattached to a solid support. If a particle is attached to the solidsupport via a complex that comprises an elongated region (as shown at(6)), the particle moves a distance (8) that is a function of the lengthof the probe. If the particle is non-specifically bound to the solidsurface (as shown at (7)), the particle does not move or moves adistance significantly less than the specifically-bound particle (6).

FIG. 4 depicts an embodiment wherein the first probe comprising anelongated region (1) and the second probe comprising an elongated region(2) are exposed to a sample under conditions such that if the targetanalyte (3) is present in the sample then it binds to the first probe(1) and the second probe (2). Upon exposure of a particle (4) to thesample, the particle (4) couples to the first probe (1). Upon exposureto a solid support (5), the second probe (2) couples to the solidsupport (5).

FIG. 5A depicts an embodiment wherein the first (1) and second (2)probes are capable of binding both the target analyte (3) and a secondmolecule (9), and a rotational force is applied to the complex which mayresult in detectable coiling or supercoiling of the complex.

FIG. 5B depicts an embodiment wherein the first probe comprises anelongated circular double stranded DNA (1). The circular double strandedDNA has a region which is single stranded where one of the strands isdiscontinuous (10). Target (3) binding to the first probe (1) bridgesthe discontinuous strand, and a rotational force is applied to thecomplex which may result in detectable coiling or supercoiling of thecomplex. The second probe (2) binds to the target and couples to thesolid support.

FIG. 5C depicts an embodiment wherein the first probe comprises anelongated circular double stranded DNA (1) having one or morefluorescent labels (11). The circular double stranded DNA has a regionwhich is single stranded where one of the strands is discontinuous (10).Target (3) binding to the first probe (1) bridges the discontinuousstrand, and a rotational force is applied to the complex which mayresult in detectable supercoiling of the complex. The second probe (2)binds to the target and couples to the solid support.

FIG. 6A shows histograms of bead displacement in experiments with 44femtoMolar (fM) (top), 4.4 fM (middle) and no target (bottom). Thesehistograms were generated using the embodiment of example 3. A firstposition for each bead was determined in images taken without flow andthen a second position for each bead was determined in images taken withflow. Bead displacement is the distance between the first and secondpositions. In this embodiment, beads bound to a complex and coupled tothe solid support via the second probe, which indicates target presence,are displaced by flow and form the peak on the right (12). Beads thatare not displaced by flow form a peak on the left (13) and correspond tobeads attached to the glass via a non-specific interaction.

FIG. 6B shows histograms of bead displacement in experiments with 1picoMolar (pM) (top), 44 fM (middle) and no target (bottom). Thesehistograms were generated using the embodiment of example 3. A firstposition for each bead was determined in images taken with flow in onedirection and then a second position for each bead was determined inimages taken with flow in the opposite direction. Bead displacement isthe distance between the first and second positions. In this embodiment,beads bound to a complex and coupled to the solid support via the secondprobe, which indicates target presence, are displaced by flow and formthe peak on the right (12). Beads that are not displaced by flow form apeak on the left (13) and correspond to beads attached to the glass viaa non-specific interaction.

FIG. 7 depicts an embodiment for detection of bacterial cells, such asthose causing tuberculosis in humans. In this example, the 23S ribosomalRNA (3) of the bacteria is detected using a first (1) and a second probe(2), each complementary to a different 30 nt sequence in the RNAmolecule. The figures show particles functionalized with DNAoligonucleotides (4); a first probe (1) comprising an elongated regionof about 9,000 DNA base pairs and 30 nucleotides overhangs at each end,one overhang complementary to the DNA oligonucleotides in the surface ofthe particles (4), the other overhang complementary to the targetanalyte (3); a second probe (2) comprising a DNA oligonucleotide, onepart complementary to the target analyte (3) and another partcomplementary to capture probes for coupling to the glass substrate (5);and a glass substrate functionalized with capture probes (5). A sample,such as sputum or blood, containing the bacteria to be detected (14) issubjected to a process that lyses the cells, liberating their nucleicacids. The first probe (1), second probe (2) and particles (4) areexposed to the sample, so that a complex comprising the first and secondprobes and the target couples to a particle. The particle is flowed intoa capillary tube where the second probe hybridizes to capture probes onthe glass substrate. Images are taken in the absence and in the presenceof fluid flow. Fluid flow generates a drag force (15) which displacesthe particles that are tethered by a complex by about 3 μm.

FIG. 8A depicts a spherical particle (4) of radius a tethered to thesolid support (5) by a tether of length L. The particle experiences anhorizontal force parallel to the surface of the solid support (F) whichinduces a tension on the complex (T).

FIG. 8B depicts the ratio of the tension and horizontal force (T/F) as afunction of the ratio of tether length and particle radius (L/a) for thephysical situation depicted in FIG. 8A.

DETAILED DESCRIPTION OF THE INVENTION

It should be appreciated that the particular implementations shown anddescribed herein are examples and are not intended to otherwise limitthe scope of the application in any way.

The published patents, patent applications, websites, company names, andscientific literature referred to herein are hereby incorporated byreference in their entirety to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.Any conflict between any reference cited herein and the specificteachings of this specification shall be resolved in favor of thelatter. Likewise, any conflict between an art-understood definition of aword or phrase and a definition of the word or phrase as specificallytaught in this specification shall be resolved in favor of the latter.

As used in this specification, the singular forms “a,” “an” and “the”specifically also encompass the plural forms of the terms to which theyrefer, unless the content clearly dictates otherwise. The terms “about”and “substantially” are used herein to mean approximately, in the regionof, roughly, or around. When the terms “about” and “substantially” areused in conjunction with a numerical range, it modifies that range byextending the boundaries above and below the numerical values set forth.In general, the terms “about” and “substantially” are used herein tomodify a numerical value above and below the stated value by a varianceof less than about 20%.

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skill in the art to which the present applicationpertains, unless otherwise defined. Reference is made herein to variousmethodologies and materials known to those of skill in the art. Standardreference works setting forth the general principles of recombinant DNAtechnology include Sambrook et al., “Molecular Cloning: A LaboratoryManual,” 2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989);Kaufman et al., Eds., “Handbook of Molecular and Cellular Methods inBiology in Medicine,” CRC Press, Boca Raton (1995); and McPherson, Ed.,“Directed Mutagenesis: A Practical Approach,” IRL Press, Oxford (1991),the disclosures of each of which are incorporated by reference herein intheir entireties.

The terms “target analyte” or “analyte,” are used herein to denote themolecule to be detected in the test sample. According to the invention,there can be any number of different target analytes in the test sample(from one to one thousand, or even more). The target analyte can be anymolecule for which there exists a naturally or artificially preparedspecific binding member. Examples of target analytes include, but arenot limited to, a nucleic acid, oligonucleotide, DNA, RNA, protein,peptide, polypeptide, amino acid, antibody, carbohydrate, lipid,hormone, steroid, toxin, vitamin, any drug administered for therapeuticand illicit purposes, a bacterium, a virus, cell, as well as anyantigenic substances, haptens, antibodies, metabolites, water pollutants(such as nitrates, phosphates, heavy metals, etc.) and molecules havingan odor, such as compounds containing sulfur and/or nitrogen, forexample hydrogen sulfide, ammonia, amines, etc., and combinationsthereof.

In a preferred embodiment, the target analyte is a nucleic acid. Thenucleic acid can be from any source in purified or unpurified formincluding DNA (dsDNA and ssDNA) and RNA, including tRNA, mRNA, rRNA,siRNA, mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA-RNAhybrids, or mixtures thereof, genes, chromosomes, plasmids, the genomesof biological materials, including microorganisms such as bacteria,yeast, viruses, viroids, molds, fungi, plants, animals, humans, andfragments thereof. The nucleic acid can be single stranded DNA obtainedby exposing double stranded DNA to an exonuclease enzyme, such asexonuclease III. The target analyte can be obtained from variousbiological materials by procedures well known in the art.

In another preferred embodiment, the target analyte is a short nucleicacid containing less than about 200 base pairs or less than about 200nucleotides. In general, such molecules are difficult to detect usingPCR-based techniques because suitable primers often cannot be found insuch a short sequence. A particular case of small DNA molecules aremolecules of less than about 40 nucleotides. These molecules are smallerthan the combined size of standard PCR primers (each primer about 20nucleotides). Short nucleic acid molecules are common in nature,exemplary cases are small interfering RNA (siRNA), micro-RNA (miRNA) andits precursors, pri-miRNA and pre-miRNA, and fragmented DNA moleculesproduced after cell death and present in blood, urine and other bodyfluids.

The term “probe” is understood herein to mean one or more molecules thatare capable of binding to the target analyte and also being coupled to,depending on the context, either a solid support or a particle. Probeshave a region capable of binding to the target analyte. The term “firstprobe” is understood herein to mean the probe that is capable ofcoupling to a particle. The term “second probe” is understood herein tomean the probe that is capable of coupling to the solid support. Forexample, if the target analyte is a nucleic acid, oligonucleotide, DNA,or RNA, the region capable of binding the target analyte in both thefirst and second probe may comprise a nucleic acid, oligonucleotide,DNA, or RNA molecule having a sequence complementary to the targetanalyte and capable of hybridizing thereto. As another example, if thetarget analyte is a protein, peptide, polypeptide, or amino acid, theregion capable of binding the target analyte in both the first andsecond probe may comprise an antibody or an antigen-binding fragmentthat specifically binds to the target analyte.

The terms “coupling”, “to couple” and “coupled” refer to a covalent ornon-covalent bond between a probe and a surface, or between a probe andanother molecule covalently or non-covalently linked to the surface. Theterms “binding,” “binds,” or “bound” refer to a covalent or non-covalentinteraction between a probe and a target analyte. In either case,non-covalent interactions could be, for example, ionic, via hydrogenbonding, etc.

The term “antibody” as used in this disclosure refers to animmunoglobulin or an antigen-binding fragment thereof.

The term “antigen-binding fragment” refers to a part of an antibodymolecule that comprises amino acids responsible for the specific bindingbetween antibody and antigen. For certain antigens, the antigen-bindingdomain or antigen-binding fragment may only bind to a part of theantigen. The part of the antigen that is specifically recognized andbound by the antibody is referred to as the “epitope” or “antigenicdeterminant.” Antigen-binding domains and antigen-binding fragmentsinclude Fab (Fragment antigen-binding); a F(ab′)₂ fragment, a bivalentfragment having two Fab fragments linked by a disulfide bridge at thehinge region; Fv fragment; a single chain Fv fragment (scFv) see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883); a Fd fragment having the two V_(H)and C_(H)1 domains; dAb (Ward et al., (1989) Nature 341:544-546), andother antibody fragments that retain antigen-binding function. The Fabfragment has V_(H)-C_(H)1 and V_(L)-C_(L) domains covalently linked by adisulfide bond between the constant regions. The F_(v) fragment issmaller and has V_(H) and V_(L) domains non-covalently linked. Toovercome the tendency of non-covalently linked domains to dissociate, ascF_(v) can be constructed. The scF_(v) contains a flexible polypeptidethat links (1) the C-terminus of V_(H) to the N-terminus of V_(L), or(2) the C-terminus of V_(L) to the N-terminus of V_(H). A 15-mer(Gly₄Ser)₃ peptide may be used as a linker, but other linkers are knownin the art. These antibody fragments are obtained using conventionaltechniques known to those with skill in the art, and the fragments areevaluated for function in the same manner as are intact antibodies.

The term “elongated region” refers to a section of the complex formed bythe target analyte and the first and second probes that is sufficientlylong such that when the complex tethers a particle to a solid supportthe displacement of the particle can be detected and differentiated fromthe displacement of particles that are non-specifically attached to thesolid support. In preferred embodiments, the elongated region is abiomolecule, such as a polysaccharide, polypeptide or nucleic acid,between about 0.15 and about 20 μm long. In even more preferredembodiments, the elongated region is a double stranded nucleic acid,between about 0.5 and about 5 μm long.

The terms “test sample” or “sample” are used interchangeably herein andinclude, but are not limited to, biological samples that can be testedby the methods of the present invention described herein and includehuman and animal body fluids such as whole blood, serum, plasma,cerebrospinal fluid, urine, lymph fluids, and various externalsecretions of the respiratory, intestinal and genitourinary tracts,tears, saliva, milk, white blood cells, myelomas and the like,biological fluids such as cell culture supernatants, fixed tissuespecimens and fixed cell specimens, PCR amplification products or apurified product of one of the above samples. A “sample” may includegaseous mediums, such as ambient air, chemical or industrialintermediates, chemical or industrial products, chemical or industrialbyproducts, chemical or industrial waste, exhaled vapor, internalcombustion engine exhaust, or headspace vapor such as vapor surroundingfoods, beverages, cosmetics, vapor surrounding plant or animal tissueand vapor surrounding a microbial sample. Another example of “sample”relevant to this invention is a liquid solution produced by dissolvingmaterial collected by filtering a gaseous sample or a liquid solutionproduced by exposing the liquid to a gaseous sample. Additional samplemediums include supercritical fluids such as supercritical CO₂extricate. Other exemplary mediums include liquids such as water oraqueous solutions, oil or petroleum products, oil-water emulsions,liquid chemical or industrial intermediates, liquid chemical orindustrial products, liquid chemical or industrial byproducts, andliquid chemical or industrial waste. Additional exemplary sample mediumsinclude semisolid mediums such as animal or plant tissues, microbialsamples, or samples containing gelatin, agar or polyacrylamide.

The term “solid support” is used herein to denote any solid materialsuitable for coupling to a probe and which is amenable to the detectionmethods disclosed herein. The number of possible suitable materials islarge and would be readily known by one of ordinary skill in the art.

The term “particle” is used to indicate any solid object or fluorescentmolecule suitable for coupling to a probe and which is amenable to thedetection methods disclosed herein.

The term “surface” or “surfaces” is used to indicate the external layerof the solid support and the particles.

In exemplary embodiments, the solid support or the particles may becomposed of modified or functionalized glasses, inorganic glasses,plastics, including acrylics, polystyrene and copolymers of styrene,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon,polysaccharides, nylon or nitrocellulose, resins, and other polymers,carbon, metals, ceramics, silica or silica-based materials includingsilicon and modified silicon and silicon wafers. In aspects, the surfacecan be a composite material.

Surfaces can be functionalized with molecules by physical or chemicaladsorption. In preferred embodiments, the surfaces are functionalizedwith probes or with molecules capable of coupling to probes. Suchmethods of functionalization are known in the art. For instance, a goldsurface can be functionalized with nucleic acids that have been modifiedwith alkanethiols at their 3′-termini or 5′-termini. See, for example,Whitesides, Proceedings of the Robert A. Welch Foundation 39thConference On Chemical Research Nanophase Chemistry, Houston, Tex.,pages 109-121 (1995). See also Mucic et al., Chem. Commun. 555-557(1996) (describes a method of attaching 3′ thiol DNA to flat goldsurfaces; this method can be used to attach oligonucleotides tonanoparticles). The alkanethiol method can also be used to attacholigonucleotides to other metal and semiconductors. Other functionalgroups for attaching oligonucleotides to solid surfaces includephosphorothioate groups (see, e.g., U.S. Pat. No. 5,472,881 for thebinding of oligonucleotide-phosphorothioates to gold surfaces),substituted alkylsiloxanes (see, e.g. Burwell, Chemical Technology, 4,370-377 (1974) and Matteucci and Caruthers, J. Am. Chem. Soc., 103,3185-3191 (1981) for binding of oligonucleotides to silica and glasssurfaces, and Grabar et al., Anal. Chem., 67, 735-743 for binding ofaminoalkylsiloxanes and for similar binding of mercaptoaklylsiloxanes)-.Oligonucleotides terminated with a 5′ thionucleoside or a 3′thionucleoside may also be used for attaching oligonucleotides to solidsurfaces. Another example of surface functionalization that is importantfor the present invention is the immobilization of antibodies and otherbinding members to the surface either by physical adsorption or bydirect or indirect chemical linkage. For instance, surfaces can befunctionalized by chemically linking streptavidin molecules to them,which are capable of coupling to probes comprising one or more biotinmolecules. The following reference describes the attachment of biotinlabeled oligonucleotides to a streptavidin functionalized surface. Shaiuet al., Nucleic Acids Research, 21, 99 (1993). Digoxigenin andanti-Digoxigenin antibodies can also be used to attach probes tosurfaces.

The surfaces can be functionalized by a monolayer of one or moremolecules. Methods of producing self-assembled monolayers are well knownin the art. In particular, there are several known methods to assemblemonolayers of thiolates on metal surfaces. See e.g., Love, J. C. et al.,Chem. Rev., 105, 1103 (2005).

The surface functionalization methods described above can be used tocouple molecules that prevent or reduce non-specific interactions withthe surface. For instance, after immobilization on to the surface of ananalyte binding molecule, such as a ssDNA or an antibody, physicaladsorption on the surface of a protein that blocks non-specificinteractions is often conducted. Common proteins used as blockers are:bovine serum albumin (BSA), fish serum and milk proteins, such ascasein.

The following references describe other methods that may be employed toattach oligonucleotides to surfaces: Nuzzo et al., J. Am. Chem. Soc.,109, 2358 (1987) (disulfides on gold); Allara and Nuzzo, Langmuir, 1, 45(1985) (carboxylic acids on aluminum); Allara and Tompkins, J. ColloidInterface Sci., 49, 410-421 (1974) (carboxylic acids on copper); Iler,The Chemistry Of Silica, Chapter 6, (Wiley 1979) (carboxylic acids onsilica); Timmons and Zisman, J. Phys. Chem., 69, 984-990 (1965)(carboxylic acids on platinum); Soriaga and Hubbard, J. Am. Chem. Soc.,104, 3937 (1982) (aromatic ring compounds on platinum); Hubbard, Acc.Chem. Res., 13, 177 (1980) (sulfolanes, sulfoxides and otherfunctionalized solvents on platinum); Hickman et al., J. Am. Chem. Soc.,111, 7271 (1989) (isonitriles on platinum); Maoz and Sagiv, Langmuir, 3,1045 (1987) (silanes on silica); Maoz and Sagiv, Langmuir, 3, 1034(1987) (silanes on silica); Wasserman et al., Langmuir, 5, 1074 (1989)(silanes on silica); Eltekova and Eltekov, Langrnuir, 3, 951 (1987)(aromatic carboxylic acids, aldehydes, alcohols and methoxy groups ontitanium dioxide and silica); Lec et al., J. Phys. Chem., 92, 2597(1988) (rigid phosphates on metals).

As used herein, a “detectable signal” which can be generated accordingto the invention includes, but is not limited to, an electrical,mechanical, optical, acoustic or thermal signal. In preferredembodiments, the detectable signal is optical or electrical.

As used herein, a “polymer” is a molecule formed by monomers in whicheach monomer is covalently linked to other monomers.

The term “monomer” is used herein to refer to a molecule that has theability to combine with identical or other molecules in a process knownas polymerization. The polymerization reaction may be a dehydration orcondensation reaction (due to the formation of water (H₂O) as one of theproducts) where a hydrogen atom and a hydroxyl (—OH) group are lost toform H₂O and an oxygen molecule bonds between each monomer unit.

The term “monomer” includes any chemical group that can be assembledinto a polymer. A wide variety of monomers may be used for synthesizinga polymer. For example, a polymer of the invention may be composed ofmonomers that have hydrophilic groups, and/or hydrophobic groups pendantfrom their backbones. Accordingly, a polymer may include side chains “R”pendant from a structurally repetitive backbone. Exemplary backboneswith side chains include:

—(CO—N(—R)—CH₂)—;

—(O—Si(—CH₃)(—R))—;

—(CH₂—CH(—R)—CO—NH)—;

—(CH₂—CH(—R)—O)—; and

—(CH₂—C₆H₄—CO—N(—R))—.

—(CH₂—CHR)—, or —(CH₂—CH₂—CHR)—;

—(CF₂—CFR), or —(CF₂—CF₂—CFR)—; and

—(CH₂—CH(—CO—NHR))—.

Examples of polymers suitable for use in this invention are polyethyleneoxide (PEO), polyethylene glycol (PEG), polyisopropylacrylamide(PNIPAM), polyacrylamide (PAM), polyvinyl alcohol (PVA),polyethylenimine (PEI), polyacrylic acid, polymethacrylate andpolyvinylpyrrolidone (PVP) polyvinyls, polyesters, polysiloxanes,polyamides, polyurethanes, polycarbonates, fluoropolymers, polyethylene,polystyrene, polybutadiene, polydimethylsiloxane (PDMS), polypropylene,polymethylmethacrylate, polytetrafluoroethylene and polyvinyl chloride(PVC).

Additional examples of suitable polymers include, but are not limitedto, those described in the references cited in this written descriptionand incorporated by reference herein. Nomenclature pertinent tomolecular structures, as well as description of monomers and side chainstructures useful for the present invention can be found in U.S. PatentPublication No. U.S. 2009/0011946, which is hereby incorporated byreference in its entirety.

As used herein, the term “polysaccharides” refers to polymericcarbohydrate structures, formed of repeating units (either mono- ordi-saccharides) joined together by glycosidic bonds. Polysaccharides ofthe invention are preferably linear, but may contain various degrees ofbranching. Additionally, polysaccharides are generally heterogeneous,containing slight modifications of the repeating unit. Examples ofpolysaccharides suitable for the invention include homopolysaccharidesor homoglycans, where all of the monosaccharides in a polysaccharide arethe same type, and heteropolysaccharies or heteroglycans, where morethan one type of monosaccharide is present. In exemplary embodiments,the polysaccharide is a starch, glycogen, cellulose, or chitin.

Polysaccharides of the invention have the general formula ofC_(x)(H₂O)_(y). In some embodiments, X is about 100 to about 100,000,about 200 to about 10,000, about 500 to about 5,000, or about 1,000 toabout 2,000. In another embodiment, polysaccharides have repeating unitsin the polymer backbone of about six-carbon monosaccharides and can berepresented by the general formula of (C₆H₁₀O₅)_(n) where n is about 30to about 100,000, about 200 to about 10,000, about 500 to about 5,000,or about 1,000 to about 2,000.

As used herein, the terms “polynucleotide,” “oligonucleotide,” “nucleicacid” and “nucleic acid molecule” are used interchangeably herein torefer to a polymeric form of nucleotides of any length, and may compriseribonucleotides, deoxyribonucleotides, analogs thereof, or mixturesthereof. This term refers only to the primary structure of the molecule.Thus, the term includes triple-, double- and single-strandeddeoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”) or RNA/DNA hybrids. It alsoincludes modified, for example by alkylation, and/or by capping, andunmodified forms of the polynucleotide. More particularly, the terms“polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acidmolecule” include polydeoxyribonucleotides (containing2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), includingtRNA, rRNA, siRNA, and mRNA, whether spliced or unspliced, any othertype of polynucleotide which is an N- or C-glycoside of a purine orpyrimidine base, and other polymers containing nonnucleotidic backbones,for example, polyamide (e.g., peptide nucleic acids (PNAs)) andpolymorpholino (commercially available from the Anti-Virals, Inc.,Corvallis, Oreg., as Neugene) polymers, and other synthetic nucleic acidpolymers providing that the polymers contain nucleobases in aconfiguration which allows for base pairing and base stacking, such asis found in DNA and RNA. The term nucleotides include hybrids thereof,for example between PNAs and DNA or RNA, and also include known types ofmodifications, for example, labels, alkylation, “caps,” substitution ofone or more of the nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoramidates, carbamates,etc.), with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalkylphosphoramidates, aminoalkylphosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingenzymes (e.g. nucleases), toxins, antibodies, signal peptides,poly-L-lysine, etc.), those with intercalators (e.g., acridine andpsoralen), those containing chelates (e.g., metals, radioactive metals,boron, oxidative metals, etc.), those containing alkylators, those withmodified linkages (e.g., alpha anomeric nucleic acids, etc.).

Oligonucleotides of defined sequences are used for a variety of purposesin the practice of the invention. Methods of making oligonucleotides ofa predetermined sequence are well-known. See, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein(ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press,New York, 1991). Solid-phase synthesis methods are preferred for botholigoribonucleotides and oligodeoxyribonucleotides (the well-knownmethods of synthesizing DNA are also useful for synthesizing RNA).Oligoribonucleotides and oligodeoxyribonucleotides can also be preparedenzymatically.

As used herein, the term “polypeptides” refers to a polymer formed fromthe linking, in a defined order, of preferably, a-amino acids, D-,L-amino acids and combinations thereof. The terms “peptides,”“oligopeptides,” and “proteins” are included within the definition ofpolypeptide. The term includes polypeptides containingpost-translational modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations, and sulphations. Inaddition, protein fragments, analogs (including amino acids not encodedby the genetic code, e.g. homocysteine, ornithine, D-amino acids, andcreatine), natural or artificial mutants or variants or combinationsthereof, fusion proteins, derivatized residues (e.g. alkylation of aminegroups, acetylations or esterifications of carboxyl groups) and the likeare included within the meaning of polypeptide. The link between oneamino acid residue and the next is referred to as an amide bond or apeptide bond. The terms do not refer to a specific length of thepolypeptide.

In some embodiments, the elongated region of one or both probes comprisea non-biological hydrophilic polymer, such as polyethylene oxide (PEO),polyethylene glycol (PEG), polyisopropylacrylamide (PNIPAM),polyacrylamide (PAM), polyvinyl alcohol (PVA), polyethylenimine (PEI),polyacrylic acid, polymethacrylate and polyvinylpyrrolidone (PVP), or acombination thereof.

In preferred embodiments, the elongated region of one or both probesand/or the target analyte comprises a biomolecule, such as apolysaccharide, polynucleotide, or a polypeptide, or a combinationthereof.

A preferred embodiment is a method of detecting a target analyte in asample. In this method, a complex is provided, the complex formed from:a first probe coupled to a particle and bound to said analyte ifpresent, and a second probe coupled to a solid support and bound to saidanalyte if present, so that if the target analyte is present in thesample, the particle is indirectly coupled to the solid support via acomplex formed by the target analyte and the first and second probes,and wherein the complex comprises an elongated region. It will beunderstood that the provided complex may have been formed in anypossible manner and order of steps. It will also be clear that theperson(s) who carries out any of the complex-formation steps may, butneed not be, the person who performs the subsequent steps in theprocess, i.e., applying the force or measuring the Brownian motion.

Preferably, the first probe, the second probe, and the target analytecomprise nucleic acids or oligonucleotides. More preferably, the firstprobe and second probe each comprise a region for binding the targetanalyte. The first probe couples to a particle at a different locationthan where the target analyte binds to the first probe. The second probecouples to the solid support at a different location than where thetarget analyte binds to the second probe. The complex formed by thetarget analyte and the first and the second probe comprises an elongatedregion. In a preferred embodiment, the elongated region of the complexformed by the target analyte and the first and the second probecomprises double-stranded DNA having a total length ranging from about500 base pairs to about 60,000 base pairs, for example, from about 1,500base pairs to about 15,000 base pairs.

In preferred embodiments, if the target analyte is a nucleic acid, thenucleic acid target can form base pairs with unpaired nucleotides in thefirst probe and with unpaired nucleotides in the second probe. In anaspect of this embodiment, the first probe further comprises a regionfor coupling to the particle. For example, the first probe can comprisea protein, a peptide, or an antigen covalently attached to the 5′ or 3′end of a nucleic acid.

In another preferred aspect, the target analyte is not a nucleic acid.When the target analyte is not a nucleic acid, the first and secondprobes preferably comprise an antibody.

In yet another preferred aspect, the target analyte is a nucleic acidand the first and second probes bind to locations on the target that areat least 500 nucleotides from each other. According to this aspect thetarget can be either double or single stranded. When the target issingle stranded, the force required to extend it is significantly higherthan the force required to extend a double stranded nucleic acid(Current Opinion in Structural Biology 2000, 10:279; Nucleic AcidsResearch 2014 (42), 3:2064). The force required to extend the singlestranded nucleic acid can be modified by changing solution properties,such as ionic strength and temperature, and/or adding a molecule thatbind to the single strand.

When the target analyte is a nucleic acid molecule, exposure of thetarget analyte to the first and/or second probe is preferably conductedunder high stringency conditions. High stringency conditions favor thehybridization of nucleic acid molecules which are perfectlycomplementary or substantially perfectly complementary to singlestranded nucleic acids in the probe and make more unlikely the bindingof targets which are not perfectly complementary or substantiallyperfectly complementary. After exposure of the target solution to thefirst and/or second probe, washing or exposing the probes to a mediumwith high stringency can remove non-perfectly complementary molecules aswell. High stringency conditions occur at high temperature, low saltconcentration and high pH. Also the presence of certain chemicals, suchas formamide, can increase the stringency of the solution. In anembodiment, exposure of the target to probes and washing, whenperformed, are conducted preferably at temperatures between 20° C. and70° C., ionic strength between 0.01 M and 1 M, and pH between 7 and 8.

Some methods of this invention contain “exposing” steps where theprobe(s), particles, and/or solid support are exposed to the sample orone another. These exposing steps can occur in any order, or evensimultaneously. For example, in one embodiment, reactants are exposed inthe following order, before applying force to the particle and measuringdisplacement: a) the first and second probe are exposed to the targetanalyte, b) the second probe which comprises a first end for coupling tothe solid support, is exposed to the solid support, c) the first probewhich comprises a first end for coupling to the particle is exposed tothe particle. If these steps are conducted under conditions that allowreactants to bind or couple and if the target analyte is present in thesample, the particle is indirectly coupled to the solid support via acomplex formed by the target, and the first and second probes. However,in another embodiment of the present invention, the steps are conductedin reverse order (c-b-a). If these steps are conducted under conditionsthat allow reactants to bind or couple and if the target analyte ispresent in the sample, the particle is indirectly coupled to the solidsupport as before: via the first probe, the target analyte and thesecond probe. In another exemplary embodiment all the steps can beconducted simultaneously. If this single step is conducted underconditions that allow reactants to bind or couple and if the targetanalyte is present in the sample, the particle is indirectly coupled tothe solid support in the same manner as the previous two examples: viathe first probe, the target analyte and the second probe. Any orderand/or combination of steps that are conducted simultaneously will havethe same result, if each step is conducted under conditions that allowreactants to bind or couple.

In preferred embodiments, the particle is a solid bead with a diameterbetween about 0.1 μm and about 20 μm, or between about 0.3 μm and about5 μm. Preferred bead materials are: silica-based glasses, such as quartzand borosilicate; zirconium and organic polymers, such as polystyrene,melamine resin and polyacrylonitrile.

In preferred embodiments, the particle is a superparamagnetic bead witha diameter between about 0.5 μm and about 5 μm. Superparamagnetic beadsare commonly used in biotechnological applications. They consist of apolymer matrix that contains small (<about 10 nm) particles of aferromagnetic material in it. The small size of the ferromagneticparticles makes them superparamagnetic. As a result, the beads aremagnetic only under the influence of an external magnetic field.

In some embodiments, the particle is a quantum dot. A quantum dot istypically less than about 10 nm and made of semiconductor materials thatdisplay quantum mechanical properties. As a result of these properties,the electronic characteristics of quantum dots are related to the sizeand shape of the individual crystal. Quantum dots are fluorescent andthe emission frequency increases as the size of the quantum dotdecreases. Therefore the color of quantum dots can be controlled bytheir size.

In some embodiment, the particle is a nanorod. Preferably, the length ofthe nanorods is at least about 0.5 μm. Methods of making nanorods ornanowires are known in the art. See for example, Hahm and Mieber, NanoLett, 4, 51-54 (2004) (silicon nanorods); Li et al., Appl. Phys. Lett.4, 4014-1016 (2003) (In2O3 nanorods); Liu et al., Phys. Ev. B. 58,14681-14684 (1998) (Bismuth nanorods); Sun et al., Appl. Phys. Lett. 74,2803 (1999) (Nickel nanorods); Ji et al., J. Electrochem. Soc. 150,C523-528 (2003) (Au/Ag multilayers and multisegment nanorods); Celedonet al., Nano Lett., 9, 1720-1725 (2009) (Pt/Ni multisegment nanorods);O'Brien et al., Adv. Mater. 18, 2379-2383 (2006) (polymer nanorods); Liuet al. Nanotechnology 20, 415703 (2009) (superparamagnetic andferromagnetic Ni nanorods).

In some embodiments, the particle comprises a fluorescent molecule.These molecules are known to those skilled in the art. For example, afluorescent nucleic acid can be created in a PCR reaction where one ofthe deoxynucleotides in the reaction mix has a florescent label. Acommonly used labeled deoxyadenosine triphosphate for this procedure isFluorescein-12-dATP. Protocols to label nucleic acid molecules arereadily available (Nucl. Acids Res. (1994) 22 (16):3418; Nat Biotechnol.(2008) 26(3):317; Nat Biotechnol. (2000) 18 (2):233). Another example offluorescent molecule is a single fluorophore, such as Cy3 and othercyanines, and fluorescein.

In some embodiments, the probes and/or target may be labeled before orafter the application of force with at least two particles, one particleat one end of the probe-target complex, the other at the other end. Inone embodiment, the elongated region of the complex may be labeledsubstantially along its length with fluorescent molecules. For example,the elongated region may be a double stranded DNA that is labeled with anucleic acid fluorescent dye, such as YOYO-1. The approximate length ofthe elongated region can be determined from the position of saidparticles after the application of force. In these embodiments thediscrimination of non-specific interaction is based on the length of theelongated region. If the full length of the elongated region isobserved, it means that the target analyte is present. Instead, if afraction of the length of the elongated region is observed, it meansthat the attachment to the solid support is via non-specificinteractions.

One embodiment of the invention is shown in FIG. 1A. According to thisembodiment, the first probe and second probes are exposed to a samplecomprising the target analyte. After the target analyte binds to boththe first and second probes, particles are introduced into the sample,which couples to the first probe. After the particle couples to thesecond probe, the sample is exposed to a solid support, where the secondprobe is coupled to the solid support. As explained elsewhere, the orderof exposing the probe to the sample, the solid support and/or theparticles can vary. Thus, for example, the particle can be exposed tothe first probe prior to exposure of the target analyte to the first andsecond probe.

Another embodiment of the invention is shown in FIG. 1B. According tothis embodiment, the second probe is exposed to a sample comprising thetarget analyte. The second probe comprises an antibody specific for thetarget analyte. After the target analyte binds to the probe, particlesare introduced into the sample. The particles are functionalized with afirst probe that comprises an antibody specific for the target analyte.After the first probe binds to the target analtye, the sample is exposedto a solid support, where the second probe is coupled to the solidsupport. As explained elsewhere, the order of exposing the probe to thesample, the solid support and/or the particles can vary. Thus, forexample, the first probe can be exposed to the target analyte prior toexposure of the target analyte to the second probe.

Other embodiments of the invention are shown in FIG. 2 . According tothese embodiments, the analyte comprises a nucleic acid and the probescomprise a nucleic acid. One probe comprises a first end or region forcoupling to either the particle or the solid support and a second end orregion for binding to the analyte, and either the particles or the solidsupport comprise a second analyte binding region. In one embodiment(FIG. 2A), the first probe (1) is covalently attached to the surface ofa particle (4). After the target analyte (3) binds to the first (1) andsecond (2) probes, the sample is exposed to a solid support (5), wherethe second probe (2) is coupled to the solid support. As explainedelsewhere, the order of exposing the probe to the sample, the solidsupport and/or the particles can vary. In another embodiment (FIG. 2B),the second probe (2) is a nucleic acid coupled to the solid support (5).The target analyte (3) can be exposed to the first probe (1) prior toexposure of the target analyte (3) to the second probe (2) or,alternatively, the target analyte can be exposed to the second probe (2)prior to exposure of the target analyte (3) to the first probe (1). Inyet another alternative, the target analyte can be exposed to the probeand solid support simultaneously. After the target analyte binds to boththe first and second probes, particles are introduced to the sample,where a particle couples to the first probe. Alternatively, the firstprobe can be coupled to the particle prior to exposure to the sampleand/or prior to exposure to the second probe.

Another embodiment of the invention is shown in FIG. 4 . According tothis embodiment, the first (1) and second (2) probes are exposed to asample comprising the target analyte (3). The target analyte can beexposed to the first probe prior to exposure of the target analyte tothe second probe or, alternatively, the target analyte can be exposed tothe second probe prior to exposure of the target analyte to the firstprobe. In yet another alternative, the target analyte can be exposed tothe first and second probes simultaneously. After the target analytebinds to both the first and second probes, particles (4) are introducedinto the sample, which couple to the first probe. After the particlecouples to the first probe, the sample is exposed to a solid support(5), where the second probe is coupled to the solid support.Alternatively, the particle can be exposed to the first probe prior toexposure of the target analyte to the first and second probe.

In an aspect according to some of the embodiments, a force is applied tothe particles. The force field acts on the particle and pulls it awayfrom its initial position (e.g. a magnetic field acting on a magneticparticle or a flow exerting a drag on a particle). The device is exposedto a sample in conditions such that if the target analyte is present,then a complex is formed by the target analyte and the first and secondprobes. Consequently, when a force is applied, the particle that isassociated with the complex will move a distance that is a function ofthe length of the complex. As shown in FIG. 3 , this amount ofdisplacement (8) indicates specific binding. Correspondingly, theparticles that are non-specifically bound (7) to the solid support willmove a shorter distance, if at all, than the specifically-boundparticles.

In one embodiment, the detection device is exposed to the sample inconditions such that the number of beads tethered by probes isproportional to the concentration of the target analyte. In this manner,the detectable signal is proportional to the concentration of the targetanalyte, thereby permitting the concentration of the target analyte inthe sample to be determined.

Preferred embodiments use probes having elongated regions of multipledifferent lengths, with each probe of a certain length having a regioncapable of binding a specific target analyte, in such a manner that eachprobe length is associated with a different target analyte. In thisembodiment, the approximate concentration of multiple target analytes ina sample can be determined in a single assay by measuring differentdisplacements of particles after application of force, grouping thembased on the amount of displacement and counting the number of particlesassociated with each possible displacement.

In related embodiments, the multiplexing capability is further increasedby modifying the probes having elongated regions in such a manner thateach of them may have more than one length and the change of length istriggered by an external agent. Examples of external agents that cantrigger a change of length are temperature, ionic strength, pH, force,an auxiliary molecule, such as a nucleic acid, an enzyme a detergent,etc. In these embodiments, the identity of a target is determined aftermeasuring the displacement of the particle before and after triggeringthe change of length. The change of length can be triggered multipletimes. For example, in an assay with 100 different probes having anelongated region, each probe specific to a different target analyte canbe created by a set of probes that have 10 different lengths beforetriggering the length change and wherein each probe experiences one outof 10 possible different length changes upon triggering the lengthchange. An example of a probe with an elongated region having a lengththat can be changed by an external agent is a probe in which two remotepositions in the probe interact in such a manner that an internal loopis formed. In this case, the external agent can trigger the release orthe formation of an internal loop. The characteristics of the externalagent required to trigger the change of length are controlled by thecharacteristics of the interaction. For example, if the interaction isthe hybridization between nucleic acid molecules, the specific sequencecan be used to modulate the characteristics of the triggering agent. Anexample of an external agent that can trigger the release of an internalloop formed by hybridization between nucleic acid molecules is anauxiliary nucleic acid complementary to one of the molecules in thehybridization region that holds an internal loop. When the probe isexposed to this auxiliary nucleic acid, the auxiliary nucleic acid candisplace one of the strands in the hybridization region, therebyreleasing the loop. Another example of an external agent is an auxiliarynucleic acid that has a first and a second region, wherein the firstregion is complementary to a first region in a nucleic acid probe andthe second region is complementary to a second region in the probe. Whenthe probe is exposed to this nucleic acid, the nucleic acid binds to thetwo regions in the probe which produces an internal loop. In relatedembodiments, the auxiliary molecules are proteins that can be usedsimilarly to the nucleic acid described above with the purpose ofreleasing or forming internal loops.

The surfaces and probes of the present invention may have a plurality ofdifferent analyte binding molecules attached to them, and as a result,the tethering of beads to the solid support could be triggered by aplurality of target analytes.

In one embodiment, a solid support may have an array of regions, eachregion comprising a second probe specific to a unique target molecule.Thus, exposure of the solid support to the sample captures differenttargets at different locations in the array. Therefore, detection ofparticle displacement in a specific array region indicates that thecorresponding target molecule is present in the sample. According tothis embodiment, a method is provided for creating a unique profile orfingerprint of a sample having any number of different target analytes(e.g., any of two through one thousand, or even more). As such, profilesfrom different samples can be stored in a database and/or compared fordiagnostic purposes for the detection of diseases or disorders.

Another embodiment uses multiple distinguishable particles, wherein eachdifferent particle comprises a different first probe specific to adifferent molecule. For example, fluorescent beads of different colorsare functionalized with different antibodies, one antibody kind for eachbead color. In this manner, the specific target molecules present in thesample are identified by detecting the color of the tethered beads thatare displaced under a force. Alternatively, beads of different sizes ordistinguishable strings of fluorescent molecules or particles can beused (Nat. Biotech. 26, 317-325, 2008).

In a preferred aspect of some of the embodiments, the applied force isfluid drag. This type of force is generated by the flow of the liquidsolution in which the particle and/or molecule is immersed. Moreprecisely, this force is applied when there is a difference between thespeed of the liquid and the speed of the particle and/or molecule. Thisforce is normally parallel to the solid support, but it can have acomponent perpendicular to the solid support if the solid support isporous. In preferred embodiments, the particles are in proximity to thesurface of the solid support and the flow is substantially parallel tothe surface of the solid support. In these embodiments, the speed of thefluid increases away from the surface of the solid support and not onlyproduces a linear force substantially parallel to the surface of thesolid support but also a torque. The terms “fluid drag” and “fluid dragforce” are used to indicate the combination of both the linear force andtorque, when it exists, experienced by the particles. In preferredembodiments, the particles have a diameter or length less than about 20micrometers and the flow is laminar, with a Reynolds number less thanabout 1. Typically, the particles and/or molecule are inside a capillarytube and flow can be generated using a pump, such as a syringe pump,connected to the capillary by a hose. Another way of generating asuitable flow is to exert an external pressure on the hose connected tothe capillary. The pressure moves a small amount of fluid into thecapillary, which displaces the particles away from the side of thecapillary where the pressure was applied. Releasing the pressure in thehose displaces the beads in the opposite direction.

In an aspect of some of the embodiments, the applied force on theparticle is fluid buoyancy. This type of force is equal to the weight ofthe fluid displaced by the particle and in the direction opposite to thegravitational force.

In an aspect of some of the embodiments, the applied force is a magneticforce. In these embodiments, the particles are magnetic, such assuperparamagnetic beads. The force is a consequence of the presence of amagnetic field which can be generated with permanent magnets, such asiron or rare earth magnets, or electromagnets. The magnetic force can beused to pull the particles away from the glass surface, in such a waythat particles tethered via an elongated region are displaced to a planehigher than the particles non-specifically attached to the surface. Thistype of displacement can be detected optically using an imaging systemable to image planes parallel to the solid support. In this imagingsystem, this type of displacement produces a change in diffractionpattern when the particle moves to a different focal plane.Alternatively, the magnetic force can be used to pull the particles in adirection parallel to the surface of the solid support. This type ofdisplacement is easily detected as a change of position of the particlesin the image using an optical system.

In another aspect of some of the embodiments, the applied force isgravitational. In these embodiments the direction of the force is alwaystoward the center of the earth and therefore its direction with respectto the solid support is determined by the orientation in space of thesolid support.

In another aspect of some embodiments, the applied force is centrifugal.In these embodiments, the particles are subjected to a motion thatchanges direction. Preferably, the motion is a rotational motion.

In another aspect of some of the embodiments, the applied force iselectrical. An electrical force is generated when at least twoelectrodes having different voltage are introduced in the solutiongenerating a voltage gradient.

In another aspect of some of the embodiments, the force is applied tothe target-probe complex using flow, a receding meniscus, or a voltagegradient.

In another embodiment, the formed complex is coupled to a substantiallyflat solid support, and is subjected to a force substantially parallelto the solid support. In that embodiment, the force is selected suchthat it removes non-specifically bound particles from the support. Then,using suitable detection methods, the presence of remaining particlesindicates the presence of the target analyte in the sample.

In embodiments that use a force substantially parallel to the solidsupport, such as embodiments that apply fluid drag substantiallyparallel to the solid support, force can remove non-specifically boundparticles while not significantly reducing the signal because being partof a complex with an elongated region reduces the force experienced bythe target analyte. When force substantially parallel to the solidsupport is applied on particles bound to the solid support, the tensionon the tether decreases with tether length (Langmuir 1996, 12(9): 2271).Therefore, non-specific interactions, which are normally tethers about10 nanometers long, experience tensions that are significantly higherthan the tension that a target bound in an elongated complex experience.This property of long tethers allows in embodiments of the presentinvention the removal of non-specifically bound particles withoutsignificantly affecting specifically bound particles. Using complexescomprising an elongated region larger than the non-specific tetherspresent in a particular assay improves the selective removal ofnon-specifically bound particles in that assay. If the tethered particleis a sphere of radius a touching the solid support that experiences aforce parallel to the solid support, as shown in FIG. 8A, then thetension (T) in the tether (length L) as a function of the horizontalforce (F) can be calculated by balancing forces and torques. FIG. 8Bshows the value of the ratio of tension and horizontal force (T/F) as afunction of the ratio of tether length and particle radius (L/a). Thetension in the tether dramatically increases for tether lengths that areless than half the radius of the particle. For example, for a tetherthat is 0.01 times the radius of the particle, the tension is 7 timeshigher than the horizontal force, while for a tether that is 2 times theradius of the particle, the tension is only 6% higher than thehorizontal force. In embodiments that apply fluid drag substantiallyparallel to the solid support, the torque applied on the particle by thedrag further increases the difference in the tension experienced byshort versus long tethers.

The application of force to the complex formed by the target analyte andthe first and second probes either directly or indirectly through forceapplied to the particle can increase the target specificity of thedetection system by removing complexes where the target is not the exactbinding partner of the binding regions in the probes. When the target isa nucleic acid, this situation takes place, in most cases, when thetarget is not perfectly complementary to a nucleic acid region in theprobes. The application of force is a novel form of hybridizationstringency. This stringency can be modulated by the configuration of thestructure formed by probes when they bind to the target. In particularfor nucleic acids, a probe can hybridize to a target in two main typesof configurations. In a first configuration, the axis of the duplex isin the direction of the force and the application of force tends todisrupt all the base pairs simultaneously. In a second configuration,the axis of the duplex is perpendicular to the direction of the forceand the application of force tends to disrupt base pair in a progressiveorder, starting with the ones closer to the point of force application.The stringency can be modulated by the amount of force applied (CurrentOpinion in Chemical Biology 2008, 12: 640, PNAS 2006 (103),16:6190).

The term “coiling” as used herein refers to a conformational changesuffered by bundles of two or more polymers subjected to a rotationalforce. A simple example of “coiling” takes place when two linearpolymers are held together by holding a first end from each polymertogether at one location and a second end of the polymers together at asecond location, if the second end of one polymer rotates around thesecond end of the other polymer, the two polymers will wind around eachother. As rotations accumulate the two polymers will start presenting amore compact conformation. This phenomenon is referred in the presentinvention as “coiling”. Another example of coiling is “supercoiling”. Inthis case, two or more nucleic acid strands are connected by basepairing and in the absence of torsional stress wind around each other.“Supercoiling” takes place when the strands are either winded orunwinded from their natural amount of winding. “Supercoiled” structureshave writhe (PNAS 1978, 75 (8) 3557).

In some embodiments, the present invention may be incorporated into anassay as described in: International Patent Publication WO 2013/059044published Apr. 25, 2013 and entitled “Detection Units and Methods forDetecting a Target Analyte”, United States Patent ApplicationPublication US 2014/0099635 A1 published Apr. 10, 2014 and U.S.Provisional Application 61/983,684, filed on Apr. 24 2014, the contentsof these publications are incorporated by reference herein. In some ofthese embodiments, and referring to present FIG. 5A, the particle may bea magnetic particle (4) and both probes may be double stranded DNAmolecules, the first probe (1) couples to the particle with at least twoattachment points and the second probe (2) couples to the solid supportwith at least two attachment points. In addition the first and secondprobes have regions to bind a second molecule (9) different from thetarget analyte (3), in such a manner that when both target and secondmolecule bind to the probes the entire complex is made of two strands,one strand contains the target and the other the second molecule. If arotational force is applied to the magnetic particle by rotating amagnetic field, torsional stress is accumulated in the complex, whichforms supercoils that dramatically shorten the end-to-end distance ofthe complex after a certain level of torsional stress is reached.Therefore, supercoiling can be readily detected as a reduction in thedisplacement of the particle (4) under an applied force or as areduction in the Brownian motion of the particle. This strategy can beused to increase the specificity and the discrimination of non-specificinteractions in the system. The torsional stress in the complex tends todisrupt mismatched targets faster than perfectly matched ones.Therefore, requiring for target detection that a bead displace first,and then the complex supercoils, imposes a second condition thatincreases target specificity. In addition, non-specific interactions canbe discriminated because non-specifically bound beads cannot supercoil.

In other embodiments, and referring to FIG. 5B, the first probe is acircular double stranded DNA molecule, wherein one of the strands has adiscontinuity in a region designated as “active segment” (10). Thecircular molecule is not capable of supercoiling because of thisdiscontinuity. The discontinued strand has flaps that have sequencecomplementary to different regions of the target analyte (3). When thetarget analyte binds (3) to the active segment (10), it bridges the twosides of the discontinued strand, which enables the circular molecule tosupercoil. If a rotational force is applied to the first probe, forexample by an intercalator molecule, torsional stress is accumulated inthe probe, which forms supercoils that dramatically change theconformation of the probe. Therefore, supercoiling can be readilydetected as a change in the displacement of the particle (4) under anapplied force or a change in the Brownian motion of the particle (4). Asin the previous embodiments, this strategy can be used to increase thespecificity and the discrimination of non-specific interactions in thesystem. In other embodiments, and referring to FIG. 5C, the first probecomprises a circular double stranded DNA molecule wherein one of thestrands has a discontinuity and the particle comprises a fluorescentmolecule (11). The first probe may have multiple fluorescent labels. Thediscontinuous strand has flaps that have sequence complementary todifferent regions of the target analyte (3). When the target analytebinds (3) to the active segment (10), it bridges the two sides of thediscontinuous strand, which enables the circular molecule to supercoil.If a rotational force is applied to the first probe, for example by anintercalator molecule, torsional stress is accumulated in the probe,which forms supercoils that dramatically change the conformation of theprobe. Therefore, supercoiling can be readily detected as a change inthe displacement of the particle under an applied force or a change inthe length of the complex under an applied force. As in the previousembodiments, this strategy can be used to increase the specificity andthe discrimination of non-specific interactions in the system.

In some embodiments of the present invention, the displacement of theparticles is detected using an imaging system, wherein the imagingsystem generates an image of the particles and/or the probes-targetcomplex that is detected by a sensing device. The image can be a regularimage or a transformed representation of the object such as a shadow.The imaging system consists of four main components: illumination,specimen, image forming part, and a detector, which are sequentiallypositioned on the spatial path. An example of an imaging system is theoptical microscope, and in this case the image forming part is thelens/lenses. Optical microscopes are well known by those of skill in theart. Optical microscopes can visualize unstained samples using imagecontrast of scattering, absorption or phase contrast, or stained sampleswith fluorescence or other scheme of light emission. The light sourceemployed in a microscope can be coherence light source (such as laser)or incoherent source (such as LED or white light source). The lenses ofa microscope can be a single lens, a series of lenses, or a compoundlens which is usually called an objective. A macroscope is anotherexample of imaging system. The main difference between a macroscope andmicroscope is the lens/objective they use. The microscope lens usuallyhas magnification equal or larger than 1×, meaning the size of image islarger than the object. That results in a small field of view. Themacroscope lens can have magnification smaller than 1×, which allows forvisualization of a large area. A lens-free imaging system is anotherexample of an imaging system. This type of system uses a digitaloptoelectronic sensor array, such as a charged coupled device (CCD) or aCMOS chip to directly sample the light transmitted through a specimenwithout the use of imaging lenses between the object and the sensorplane (Greenbaum, Nat. Methods 2012, 9, 9, 889-895; Gurkan, U. A., etal., Biotechnol. J. 2011, 6, 138-149). The lensless ultra wide-fieldcell monitoring array platform (LUCAS) (Ozcan, A. and Demirci, U. LabChip, 2008, 8, 98-106) is an example of this type of microscopes. TheLUCAS platform is based on recording the “shadow images” of microscopicobjects onto a sensor array plane. Microscopic objects are uniformlyilluminated with an incoherent light source or a laser. The cell shadowpattern is digitally recorded using a CCD or CMOS sensor array. Acoherent imaging system is another example of image system. This type ofsystem uses the object to modulate the illumination laser beam and makesthe modulated beam interfere with a reference laser beam or the sameillumination beam, then the interferential information is recorded toreconstruct the information of the object. Digital holography (Javidi,Opt. Lett., 2000, 25, 9, 610-612) and in-line holography (Xu, PNAS,2001, 98, 20, 11301-11305) are examples of this technique.

In some embodiments, the displacement of the magnetic particles isdetected from the induced current in a solenoid.

As noted above, the present invention also is directed to a kit fordetecting a target analyte in a sample, the kit comprising a) aparticle; and b) a first probe capable of binding to the analyte, and toeither a solid support or to the particle, the first probe optionallycomprising an elongated region between about 0.15 and about 20 μm long;c) packaging material; and optionally d) instructions for use. In aparticular embodiment, the kit may further comprise a second probecapable of binding to the analyte at a location different than thelocation that the first probe binds to the analyte, and which also iscapable of binding to either a solid support or to the particle. Thesecond probe may optionally contain an elongated region between about0.15 and about 20 μm long. If the first probe is for coupling to a solidsupport, the second probe is for coupling to the particle, and if thefirst probe is for coupling to the particle, the second probe is forcoupling to the solid support. The packaging material would be known toone of ordinary skill, and in certain embodiments would includeconventional bottles, vials, boxes, etc. The optional instructions foruse would preferably include conventional printed materials includedwithin the packaging material.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. In caseof conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

EXAMPLE 1

This example demonstrates a procedure to detect a target in a sample atlow concentration using magnetic beads. The first probe is generated inthe following manner A plasmid (approximately 10 kilo base pairs) islinearized using a restriction enzyme that cuts it once. Two types ofshort DNA molecules capable of being ligated to the linearized plasmidare purchased (IDT): the first short DNA molecule is labeled withDigoxigenin and the second has single stranded region complementary topart of the target. The two short DNA molecules are ligated to thelinearized plasmid. The second probe, a biotin labeled single strandedmolecule, is purchased (IDT) with a sequence complementary to part ofthe target. Super-paramagnetic beads (Life Technologies) coated withStreptavidin are purchased. The first and second probes are mixed athigh concentration (>0.01 μM) with the sample and incubated for fewminutes. Magnetic beads are added to the solution and incubated fewminutes. In order to wash unbound molecules, the solution is exposed toan external magnet that attracts the magnetic beads removing them fromthe solution. The solution is extracted from the container anddiscarded. The external magnet is taken away and the beads are suspendedin buffer solution. Beads are then flowed into a capillary tubepreviously functionalized with anti-digoxigenin proteins (Roche) and letsediment and interact with the surface of the tube. In order to washunbound beads, new buffer solution is flowed while a magnet is passedover the capillary. A large magnet is placed over the capillary to liftremnant unbound beads. The beads that remain attached to the bottom ofthe capillary are imaged using an image sensor array directly in contactwith the bottom of the capillary tube. The solution in the tube isflowed in one direction to drag the beads and then the direction of theflow is reversed to drag the beads in the opposite direction. Successiveimages of the beads during this process show that some beads move about3 micrometers in each direction while the rest of the beads bound to thesurface do not move or move significantly less. Beads that move about 3micrometers in each direction are bound to the glass surface via thecomplex of probes and target. The initial concentration of targetmolecules in the sample determines the number of these beads.

EXAMPLE 2

This example follows the same procedure as the previous example, butinstead of flowing solution in the capillary tube to distinguish thebeads tethered by probes, the Brownian motion of the beads is detected.The beads attached via the probes to the solid support undergo Brownianmotion and move within a circle of about 1 micrometer diameter. Thesebeads can be discriminated from the beads non-specifically attached tothe solid support which move significantly less.

EXAMPLE 3

This example demonstrates detection of a 60 nucleotide (nt) syntheticDNA oligonucleotide target having the sequence of a section ofMycobacterium tuberculosis rRNA. The first probe was purchased from IDTand consisted of a 24 nt single stranded oligonucleotide having asequence complementary to the 3′ end of the target and a 5′ biotinmodification. The second probe was generated in the following manner Aplasmid (8.5 kbps) was linearized using the restriction enzyme BsmB I(New England Biolabs), which cut the plasmid twice generating a largefragment (approximately 8.4 kbps/2.8 micrometers) with different 4 ntoverhangs at each end, and a small fragment which was separated anddiscarded by agarose gel purification (QIAquick Gel Extraction Kit,Qiagen). The linearized plasmid was ligated using T4 ligase to twodouble stranded DNA fragments generated by hybridizing syntheticoligonucleotides. The first fragment had one end with an overhangcompatible to one of the overhangs of the plasmid and the other end hada 30 nt overhang complementary to the 5′ end of the target. The secondfragment had one end with an overhang compatible to the other overhangof the plasmid, and the other end of the fragment had a 5′ digoxigeninmodification. The first probe was mixed with a solution containing thetarget and the temperature was raised to 65° C. for 1 minute and thenincubated at room temperature for 10 minutes. The buffer contained 800mM NaCl. A blocker oligonucleotide complementary to the first probe wasadded and incubated for 5 minutes. The second probe was added andincubated for 10 minutes. Super-paramagnetic beads (Life Technologies)coated with Streptavidin were added and incubated for 30 minutes. Themixture was then flowed into a glass capillary tube (50 mm×2 mm×0.2 mm)previously functionalized with anti-digoxigenin proteins (Roche) andbeads were let sediment for 5 minutes. A 100 mM NaCl buffer solution wasflowed to wash unbound beads. The beads that remained attached to thebottom of the capillary were imaged first in the absence of flow (45images) and then in the presence of flow (160 microliters/minute, 45images). Alternatively, the beads were imaged first with flow in onedirection (160 microliters/minute, 45 images), and then with flow in theopposite direction (160 microliters/minute, 45 images). The opticalsystem used to image the beads was composed of a LED ring light, atelecentric lens and a camera. The LED ring light provided a dark fieldillumination. The telecentric lens, which is popularly employed inmachine vision, exhibited the same magnification for objects atdifferent distances. The lens had a large depth of field of around 500micrometers. The magnification of the system was 1:1, which helped toachieve a large field of view of 6.14 mm by 4.6 mm. According to theoptical resolution of the lens, the image size of each particle wasabout 4 micrometers, which was sufficient to investigate thedisplacement of the particles. The camera used in the system had a

inch complementary metal oxide semiconductor (CMOS) chip, which had 4384by 3288 pixels with pixel size of 1.4 micrometer.

A custom code written in Matlab was used to analyze the images anddetermine the displacement of most of the beads present in the field ofview. Beads that were too close to each other (less than about 6micrometers) were not included in the analysis. The position of a beadwas defined as the average of its position in the 45 images. This systemallowed measurement of the displacement of over 3,000 beads in eachexperiment with sub-micrometer resolution. The code generated ahistogram of bead displacement (FIG. 6 ). When the first set of imageswas taken without flow (FIG. 6A), beads that moved more than 2.5micrometers where considered bound to a target-probe complex (right peakin the histogram). Beads that moved less than 2.5 micrometers wereconsidered non-specifically attached to the capillary surface. Most ofbeads that moved less than 2.5 micrometers moved less than 0.5micrometers and had on average zero displacement. These beads usuallynumbered more than 2,000. When the first set of images was taken withflow (FIG. 6B), beads that moved more than about 5 micrometers whereconsidered bound to a target-probe complex (right peak in thehistogram).

What is claimed is:
 1. A kit for detecting a target analyte in a sample,the kit for use with a solid support, comprising: a) a first probecomprising a first region configured to directly bind the target analyteat a first location and a second region configured to couple to aparticle, wherein the first probe cannot specifically couple to thesolid support in the absence of the target analyte; and b) a secondprobe comprising a first region configured to directly bind the targetanalyte at a second location different than the first location, and asecond region configured to couple to the solid support, wherein thesecond probe cannot specifically couple to the particle in the absenceof the target analyte; wherein the first probe and the second probe canbind to the target analyte, if present in the sample, to form a complexsuch that the particle can be indirectly coupled to the solid support,and wherein the length of the complex, from where the first probecouples to the particle to where the second probe couples to the solidsupport is about 1.0 micron to about 20 microns.
 2. The kit of claim 1,wherein the first probe comprises an antibody and the second probecomprises an antibody.
 3. The kit of claim 1, wherein the first probecomprises a first nucleic acid and the second probe comprises a secondnucleic acid.
 4. The method of claim 1, wherein the target analyte is aprotein.
 5. The method of claim 1, wherein the target analyte is anucleic acid molecule.
 6. A kit for detecting a target analyte in asample, the kit for use with a solid support and a detectable particle,comprising: a) a first probe comprising a first region configured todirectly bind the target analyte at a first location and a second regionconfigured to couple to a detectable particle, wherein the first probecannot specifically couple to the solid support in the absence of thetarget analyte; and b) a second probe comprising a first regionconfigured to directly bind the target analyte at a second locationdifferent than the first location, and a second region configured tocouple to the solid support, wherein the second probe cannotspecifically couple to the detectable particle in the absence of thetarget analyte; wherein at least one of the first probe, the secondprobe or a region of the target analyte between the first location andthe second location comprises an elongated region of about 1.0 micron toabout 20 microns long, and wherein the first probe and the second probecan bind to the first and second locations on the target analyte, ifpresent in the sample, to form a complex of the first probe, the secondprobe, the target analyte, and the detectable particle, where thecomplex can couple to the solid support.
 7. The kit of claim 6, whereinthe first probe comprises an antibody and the second probe comprises anantibody.
 8. The kit of claim 6, wherein the first probe comprises afirst nucleic acid and the second probe comprises a second nucleic acid.9. The kit of claim 6, wherein the elongated region is on the firstprobe.
 10. The kit of claim 6, wherein the elongated region is on thesecond probe.
 11. The kit of claim 6, wherein the elongated region is onthe portion of the analyte between the locations where the first andsecond probes bind to the analyte.
 12. The method of claim 6, whereinthe target analyte is a protein.
 13. The method of claim 6, wherein thetarget analyte is a nucleic acid molecule.
 14. A method of detecting atarget analyte in a sample comprising: a) providing a particleindirectly coupled to a solid support via a complex if the targetanalyte is present in the sample, the complex formed from: i) a firstprobe comprising a first region configured to directly bind the targetanalyte at a first location and a second region configured to couple toa detectable particle, wherein the first probe cannot specificallycouple to a solid support in the absence of the target analyte; and ii)a second probe comprising a first region configured to directly bind thetarget analyte at a second location different than the first location,and a second region configured to couple to the solid support, whereinthe second probe cannot specifically couple to the detectable particlein the absence of the target analyte; iii) the target analyte, whereinat least one of the first probe, the second probe or a region of thetarget analyte between the first and second locations comprises anelongated region of about 1.0 micron to about 20 microns long; b) eitheri) applying a force to the complex comprising the elongated region orii) measuring the Brownian motion of the particle.
 15. The method ofclaim 14, wherein the first probe comprises an antibody and the secondprobe comprises an antibody.
 16. The method of claim 14, wherein thefirst probe comprises a first nucleic acid and the second probecomprises a second nucleic acid.
 17. The method of claim 14, wherein theelongated region is on the first probe.
 18. The method of claim 14,wherein the elongated region is on the second probe.
 19. The method ofclaim 14, wherein the elongated region is on the portion of the analytebetween the locations where the first and second probes bind to theanalyte.
 20. The method of claim 14, wherein the target analyte is aprotein or a nucleic acid molecule.